or Radionuclide

Review pubs.acs.org/Organometallics

Organometallic Complexes for SPECT Imaging and/or Radionuclide Therapy Goreti Ribeiro Morais, António Paulo, and Isabel Santos* Unidade de Ciências Quı ́micas e Radiofarmacêuticas, Instituto Tecnológico e Nuclear, Instituto Superior Técnico, Universidade Técnica de Lisboa, Estrada Nacional 10, 2686-953, Sacavém, Portugal ABSTRACT: This article reviews Re and Tc organometallic chemistry and radiochemistry, focusing on the most relevant aspects to design radiotools for molecular imaging and therapy. The review comprises an overview of organometallic Re and Tc complexes with relevance to the field, particularly M(I) tricarbonyl complexes stabilized by a huge variety of ligands. These include Werner type chelators, cyclopentadienyls, carboranes, and poly(mercaptoazolyl)borates, which permitted the hitherto unprecedented use of M−C and M−H bonds. The most relevant biomolecules labeled with the different organometallic cores will also be reviewed, as well as the opportunities offered by the tricarbonyl approach to design high-performance perfusion agents.

1. INTRODUCTION Until recently, much research in organometallic chemistry has been driven by its potential for stoichiometric or catalytic transformation of organic substrates, which would otherwise be inaccessible or hardly feasible. The excellence of such research has resulted in the award of two Nobel Prizes in Chemistry, one to Y. Chauvin, R. H. Grubbs, and R. R. Schrock in 2005 and the second to R. F. Heck, E. Negishi, and A. Suzuki in 2010.1 Previously, a widespread consensus that organometallic systems were incompatible with the presence of oxygen and water, due to their distinct and enhanced reactivity, indicated little potential for these compounds in biology. However, the advent of bioorganometallic chemistry as a new research area was inspired by the fact that nature itself makes use of compounds having M−C bonds.2 Bioorganometallic chemistry now has an increasing impact in life sciences, particularly in the design of novel anticancer drugs, due to the greater variety of structural motifs and reactivity patterns offered by metal complexes in comparison to purely organic molecules. This versatility is an advantage for designing drugs with different mechanisms of action. The first encouraging results were obtained with organometallic analogues of tamoxifen, namely ferrocene derivatives, which have shown potential for breast cancer therapy.3 Since then, a variety of cyclopentadienyls or arenes of Fe, Ru, or Ti have also been synthesized and their therapeutic effects assessed.2,4 Radiopharmaceutical chemistry is another important field where organometallic compounds have a huge potential. For the large majority of metals employed in this area, e.g. lanthanides, gallium, indium, and copper, the most relevant compounds are coordination complexes.5−8 However, for Tc and Re, due to their rich chemistry, organometallic complexes have recently assumed a growing importance © 2012 American Chemical Society

through the possibility of preparing the precursors fac[M(CO)3(H2O)3]+ (M = Re, Tc) in aqueous solution.9−11 Radiopharmaceuticals are drugs containing a radionuclide in their composition. They are used in nuclear medicine for diagnosis and therapy. For diagnosis, γ- or positron-emitting radionuclides are suitable for single photon emission computed tomography (SPECT) or positron emission tomography (PET), respectively. The radiopharmaceuticals in clinical use for SPECT are predominantly metal-based compounds: the overwhelming majority are 99mTc complexes, which remains the workhorse of nuclear medicine due to its ideal nuclear properties (Table 1), low cost, and convenient availability from commercial generators. For therapy, the radiopharmaceuticals must contain radionuclides emitting ionizing particles (Auger electrons, β− or α particles) to target and damage tumor tissues. Several radiometals have nuclear decay properties suitable for therapeutic applications, such as the β−-emitting isotopes 186Re and 188Re (Table 1). The pharmacokinetics of metal-based radiopharmaceuticals can be determined by their physicochemical properties (perf usion agents) or by specific interactions (target-specif ic radiopharmaceuticals) with biological targets (Figure 1). For perfusion agents, the biological distribution is determined by blood flow and they target high-capacity systems, such as phagocytosis, hepatocyte clearance, and glomerular filtration. Specific radiopharmaceuticals target low-capacity processes, and their biodistribution is determined by specific protein interactions: e.g. enzymatic or receptor-binding interactions. Special Issue: Organometallics in Biology and Medicine Received: June 6, 2012 Published: August 1, 2012 5693

dx.doi.org/10.1021/om300501d | Organometallics 2012, 31, 5693−5714

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Table 1. Physical Properties, Production Mode, and Application of Selected Radiometals nuclide

mode of decay (%)

99m

Tc

6.0 h

ITa (100)

186

Re

89.2 h

188

Re

17 h

β− (92) ECb (8) β− (100)

I In 67 Ga 68 Ga

13.2 2.83 3.27 67.8

18

109.8 min

123 111

11 86

F C Y

90

Y 64 Cu

67

Cu Sm 166 Ho 153

a

half-life

h d d min

EC (100) EC (100) EC (100) β+ (90)

64.1 h 12.7 h

EC (10) β+ (97) EC (3) β+ (100) β+ (33) EC (66) β− (100) β− (40)

61.8 h 46.3 d 26.8 d

β+ (19) EC (41) β− (100) β− (100) β− (100)

20.3 min 14.7 h

production mode 99

application

Mo/99mTc generator 185 Re(n,γ)186Re

SPECT

188

W/188Re generator 121 Sb(α,2n)123I 111 Cd(p,n)111In 68 Zn(p,2n)67Ga 68 Ge/68Ga generator

therapy

18

PET

14 86

O(p,n)18F 11

N(p,α) C Sr(p,n)86Y

therapy

SPECT SPECT SPECT PET

PET PET

89

Y(n,γ)90Y 64 Ni(p,n)64Cu

therapy PET/ therapy

67

therapy therapy therapy

Zn(n,p)67Cu Sm(n,γ)153Sm 165 Ho(n,γ)166Ho 152

Figure 2. Sestamibi and three organometallic precursors.

developed in the 1990s some elegant and creative research at the forefront of aqueous organometallic chemistry. This research resulted in the organometallic complex fac-[99mTc(CO)3(H2O)3]+ (Figure 2).10 Unlike sestamibi, this is an excellent precursor for radiopharmaceutical development. This complex stimulated renewed interest in the design of Tc(I) organometallic radiopharmaceuticals as an alternative to classical strategies on the basis of the cores [99mTc(O)]3+, trans-[99mTcO2]+, [99mTc(N)]2+, and [99mTc-HYNIC] (HYNIC = 6-hydrazinonicotinic acid).14−16 [99mTc(NS3)X] (NS3 = tetradentate umbrella type chelator; X = unidentate C-donor coligand, preferably of the isonitrile type) and fac-[99mTc(NO)(CO)2(H2O)3]2+ represent other organometallic building blocks currently available to design radiopharmaceuticals (Figure 2).17,18 So far, these two building blocks have been much less explored than the 99mTc(I) tricarbonyl precursor. Re complexes are commonly used as surrogates for 99mTc congeners. This approach benefits from the physicochemical similarities of these two elements and avoids the use of the long-lived β− emitter 99Tc. Tc and Re can also be considered a “matched pair” suitable for obtaining radiolabeled compounds for nuclear imaging (99mTc) or radionuclide therapy (186/188Re), despite differences in the kinetics of ligand exchange reactions and redox chemistry. On the basis of isostructural Re and Tc compounds one can achieve the so-called theranostic agents, which can deliver ionizing particles (186Re/188Re) to treat a tumor or obtain images (99mTc) to make a diagnosis, overcoming undesirable differences in biodistribution and selectivity, which currently exist between distinct imaging and therapeutic tools. This paper reviews Re and Tc organometallic chemistry and radiochemistry, focusing on the aspects most relevant to the design of radiotools for molecular imaging and therapy. These include Werner chelators, cyclopentadienyls, carboranes, and poly(mercaptoazolyl)borates, which permitted the hitherto unprecedented use of M−C and M−H bonds. The most relevant biomolecules labeled with the different organometallic cores will also be reviewed, as well as the opportunities offered by the tricarbonyl approach to design high-performance perfusion agents. The present contribution aims to complement previous reviews.16,19−26 However, to provide context, some overlap is unavoidable.

IT = isomeric transition. bEC = electron capture.

Figure 1. Schematic representation of perfusion and target-specific radiopharmaceuticals.

Recently, much research has focused on the design of specific radiopharmaceuticals as new tools to image biological/ biochemical processes at the molecular level, to help diagnose or monitor underlying diseases. The design of metal-based radiopharmaceuticals usually requires the use of bifunctional chelators (BFCs), to stabilize the metal core and to couple to the bioactive molecule. The unique organometallic compound in clinical use, for decades, is sestamibi, a lipophilic cation of Tc(I) stabilized by six isonitrile ligands: [ 99m Tc(CNR) 6 ] + (R = CH 2 C(CH3)2OCH3). This radiopharmaceutical is marketed under the trademark of Cardiolite and is used as a perfusion agent for myocardium imaging (Figure 2).12,13 Together with cisplatin, sestamibi can be considered the most successful synthetic complex for medical application, from a scientific, commercial, and health care point of view. Despite its noteworthy success, sestamibi was not a good precursor to enter into the chemistry of Tc(I) in the aqueous medium. In this respect, Alberto et al. 5694


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Scheme 1. Synthesis and Aquation of Halide Re(I) and Tc(I) Tricarbonyl Complexes

Scheme 2. Aqueous Synthesis of fac-[M(CO)3(H2O)3]+ (M =

99m

Tc (3a),

188

Re (6a))

tricarbonyl precursor with 99m Tc. Initially, fac-[ 99m Tc(CO)3(H2O)3]+ (3a) was obtained by treating [99mTcO4]− (1a) with NaBH4 in the presence of CO (Scheme 2).10 However, CO is a toxic gas, unsuitable for use in hospitals and in commercial radiopharmaceutical kits. This problem was overcome using potassium boranocarbonate, K2[H3BCO2] (Figure 3), a compound previously introduced by Malone

2. ORGANOMETALLIC PRECURSORS Nonradioactive metal-based antitumoral agents can be synthesized in organic solvents and thereafter transferred to aqueous media, prior to biomedical application. In radiopharmaceutical chemistry the starting radiometals are usually available in aqueous solution and, therefore, the synthetic processes have to be performed in water. For 99mTc and 188Re the synthesis of the compounds always starts with pertechnetate or perrhenate, obtained in saline at very low concentrations, by elution of 99Mo/99mTc and 188W/188Re generators, respectively. In the 1990s, Alberto et al. started exploring synthetic methodologies for the preparation of Tc tricarbonyl complexes in aqueous solution. Initially, they showed that the halide (NEt4)2[99TcCl3(CO)3] (2) could be obtained directly from [99TcO4]− (1) by reduction with BH3 in refluxing THF saturated with CO (Scheme 1).9 The congener (NEt4)2[ReBr3(CO)3] (5) had already been reported by treating the commercially available [ReBr(CO)5] (4) with [NEt4]Br in refluxing diglyme.9,27 The halides in 2 and 5 are readily replaceable by water, affording the aquo tricarbonyl precursors fac-[M(CO)3(H2O)3]+ (M = 99Tc (3), Re (6)) (Scheme 1). More recently, the synthesis of 6 has been described by reacting 4 with water or by boiling an aqueous suspension of [Re(OTf)(CO)5].28,29 The successful synthesis of fac-[99Tc(CO)3(H2O)3]+ (3) starting from 1 was an encouraging result that prompted Alberto to study the possibility of preparing this aquo

Figure 3. ORTEP diagram of boranocarbonate anion in K(cryptand)[H3BCO2H].31

and Parry.30 The boranocarbonate reduces the Tc(VII) and acts simultaneously as a CO source, through mechanisms not yet fully understood. Currently, there is a kit formulation available for research (Isolink, Covidien), which contains sodium boranocarbonate, Na/K tartrate, sodium tetraborate decahydrate, and sodium carbonate. This formulation allows the synthesis of 3a by simple addition of Na[99mTcO4] in saline and heating at 95 °C 5695


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Scheme 3. Synthesis of Dicarbonyl−Nitrosyl Precursors

for 20 min (Scheme 2).31 Recently, Valliant et al. have shown that, using a microwave reactor and heating at 135 °C, 3a can be obtained 3 min after the addition of Na[99mTcO4] to the Isolink kit.32 Unlike 3a, the synthesis of fac-[188Re(CO)3(H2O)3]+ (6a) has not been achieved by reduction of [188ReO4]− (7) with boranocarbonate, emphasizing the differences between Tc and Re. Compound 6a was prepared by reducing 7 with a combination of K 2 [H 3 BCO 2 ] and amine−borane (BH3·NH3).11 The yield of this reaction was around 85%, and its improvement up to 97% was only achieved by running the reaction in the presence of a borohydride exchange resin (BER), used as an additional reducing agent and as an anion scavenger.33 The precursors 3/3a and 6/6a allow a versatile entry into the chemistry of Re(I) and Tc(I), as shown by the multitude of complexes reported in recent years. The d6 low-spin electronic configuration of the metal in this core provides a high kinetic stability to the complexes, leading to highly stable M−C bonds. For this reason, the three CO ligands always remain coordinated when 3/3a and 6/6a react with different ligands, which readily replace the three water molecules. Nevertheless, the water self-exchange rates in these precursors are rather slow: self-exchange rate constants (kex) are 0.49 and 0.0054 s−1 for Tc and Re, respectively.34 Such rate constants justify the need for heating to label biomolecules with fac-[M(CO)3]+ (M = 99mTc, 188Re), particularly when the ligands carrying the biomolecules are used in very low concentration and high specific activity is required for clinical application. Two organometallic precursors introduced more recently are fac-[M(NO)(CO)2(H2O)3]2+ and [M(NS3)(CNR)] (M = Tc, Re).17,18 At the macroscopic level, the complexes fac-[M(NO)(CO)2X3]− (M = 99Tc, X = Cl (8); M = Re, X = Br (10)) have been obtained by reacting the tricarbonyl precursors fac[M(CO)3X3]+ (M = Tc, X = Cl (2); M = Re, X = Br (5)) with NOHSO4 in the presence of 1 M HX. In a similar way, fac[99mTc(NO)(CO)2(H2O)3]2+ (8a) was obtained in aqueous medium (>85% yield) by treating 3a with NOHSO4 under acid conditions (Scheme 3).17,18 A priori, the three water molecules of 8a can be replaced by the same tridentate chelators that substitute those ligands in 3a, affording complexes that only differ in the overall charge. It has been claimed that this difference could be useful to improve pharmacokinetics and the

biological profile of structurally related complexes. However, the use of 8a in the design of 99mTc radiopharmaceuticals is rather rare, due to its instability at pH >7.5 and the possibility of forming different stereoisomers upon reaction with stabilizing ligands. The building blocks [M(NS3)(CNR)] (M = Tc, Re) correspond to mixed-ligand Tc(III)/Re(III) complexes containing the umbrella type ligand 2,2′,2″-nitrilotris(ethanethiol) (NS3) and isocyanides (CNR) as coligands, which preferably can carry biomolecules. A variety of biomolecules (e.g., central nervous system receptor ligands, peptides, and fatty acids) have been labeled with these building blocks within the so-called [4 + 1] mixed-ligand concept.35−37 At the macroscopic level, the building blocks [Re(NS3)(CNR)] (13) were prepared by substitution of the phosphine ligand in [Re(NS3)(PR3)] (12) by an isocyanide (Scheme 4).17,38 X-ray structural analyses of Scheme 4. Synthetic Pathways To Obtain [4 + 1] MixedLigand Complexes at Macroscopic and Tracer Levels

some of these [4 + 1] complexes confirmed that the metal is pentacoordinate with a nearly ideal trigonal-bipyramidal geometry. The trigonal plane is defined by the three sulfur atoms of the NS3 ligand, while the central nitrogen atom from the same ligand and the monodentate isocyanides occupy the apical positions. The synthesis of the radioactive congeners [M(NS3)(CNR)] (M = 188Re (13a) and 99mTc (13b)) involves in a first step the preparation of the complexes Tc(III)- and 5696


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In vivo data of complexes anchored on bi- and tridentate chelators have shown that complexes stabilized by bidentate ligands are more likely to be retained in the liver and kidneys than those anchored on tridentate chelators.42,43 It has been inferred that such differences may be related to the susceptibility of the third aqua ligand to exchange in vivo with blood proteins, resulting in less favorable biodistribution profiles and pharmacokinetics. In particular, tridentate chelators containing N-heterocyclic coordinating groups, such as pyridine, imidazole, and pyrazole, form complexes with quite favorable kinetics and stability. Examples of this type of ligand are the pyrazole-diamine introduced some years ago by the authors’ research group,48,49 histidine-based ligands explored by Alberto’s group,53 triazolecontaining compounds obtained in situ by Schibli’s group using click chemistry,52 and bis(methylpyridyl)amine derivatives reported by Valliant et al.50 (Figure 4).

Re(III)-EDTA and in a second step the replacement of EDTA by NS3 and isocyanide ligands (Scheme 4).39,40

3. COMPLEXES ANCHORED BY WERNER-TYPE LIGANDS A wide variety of bi- or tridentate chelators, particularly of the “Werner type”, have been explored to synthesize Re and Tc tricarbonyl complexes under physiological conditions.29,41−53 On the basis of tridentate chelators, several building blocks have been prepared both at macroscopic and tracer levels (Figure 4) and used to label biomolecules with high specific activity and radiochemical purity.

4. COMPLEXES ANCHORED BY “NON-CLASSICAL” LIGANDS The inherent stability and unique features of the complexes based on fac-[M(CO)3]+ (M = Re, Tc) have enabled the use of typically organometallic ligands such as bridging hydrides, cyclopentadienyls, and carboranes, previously unexplored in radiopharmaceutical chemistry. The authors have studied the coordination of dihydrobis(2mercaptoimidazolyl)borates and trihydro(2mercaptoimidazolyl)borates toward the fac-[M(CO)3]+ (M = Re, 99Tc, 99mTc) moiety under aqueous conditions. These studies led to the first Re and Tc complexes containing respectively one- or two-coordinated bridging hydrides, as confirmed by X-ray structural analysis (Scheme 6).56−58

Figure 4. Examples of 99mTc(I) tricarbonyl complexes with tridentate Werner-type ligands.29,41−52

Alberto and co-workers has also explored bidentate chelators and the so-called [2 + 1] approach for labeling biologically active molecules. These [2 + 1] complexes were obtained at macroscopic (Re/99Tc) and tracer levels (99mTc) using bidentate pyridine-2-carboxylic derivatives and imidazoles or isonitriles as monodentate coligands (Scheme 5A).54 A metal-

Scheme 6. Poly(mercaptoimidazolyl)borate Re Tricarbonyl Complexes Containing Bridging Hydridesa,56,58

Scheme 5. Examples of 99mTc(I) Tricarbonyl Complexes with Bidentate Chelators: (A) [2 + 1] Complexes; (B) Complexes with Schiff Base Ligands Obtained in Situ

a

timMe = 2-mercapto-1-methylimidazolyl.

99m

Tc complexes are formed at room temperature with relatively low ligand concentrations and are remarkably stable under physiological conditions. Such behavior shows that water and the ∼105 fold excess of Cl− present in solution do not compete with the bridging hydrides. Biodistribution studies in mice have shown that some of these complexes cross the blood−brain barrier (BBB), due to their small size, neutral charge, and lipophilicity.59 These results prompted their evaluation as building blocks for labeling brain receptor ligands, as will be discussed in section 6.

mediated reaction also allowed the in situ formation of Re/99mTc complexes stabilized by bidentate Schiff base ligands (18). These complexes were formed upon activation of coordinated aldehydes and reaction with an aliphatic or aromatic amine (Scheme 5B).55 On the basis of this result, it was claimed that such a methodology could be useful for the direct labeling of biomolecules containing free amine functions. 5697


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Despite the robustness of the B−H···Re bonds in fac-[Re{κ3H(μ-H)B(timMe)2}(CO)3] (19) and fac-[Re{κ3-H(μ-H)2B(timMe)}(CO)3] (20) under physiological conditions, reactions of 19 and 20 with a variety of monodentate and neutral substrates (e.g., imidazole, pyridine, isonitrile, and phosphine derivatives) afford mixed-ligand complexes of the [2 + 1] type.60−62 Solid-state structural analysis and NMR studies have shown that in the resulting complexes the mercaptoimidazolyl borates are coordinated in a κ2-S,S or κ2-S,H fashion, respectively, as shown for 21 and 22 in Scheme 7.

Rhenium tricarbonyl complexes anchored by hybrid poly(azolyl)borates, formed in situ, could also be obtained by reacting compound 20 with protic substrates (Scheme 8).63 Reactions of the 99mTc congeners of 19 and 20 with the neutral or protic substrates do not yield the corresponding [2 + 1] mixed-ligand complexes or complexes anchored by hybrid poly(azolyl)borates (Schemes 7 and 8). The impossibility of obtaining these complexes by reacting fac-[99mTc{κ3-H(μH)B(timMe)2}(CO)3] (19a) or fac-[99mTc{κ3-H(μ-H)2B(timMe)}(CO)3] (20a) with the appropriate substrates must be related to the low concentration of 99mTc (i.e., 10−7−10−9 M), which might render the reactions very slow, compared with those at the macroscopic level (i.e., 10−2−10−3 M). Preparation of the complex fac-[99mTc{κ3-H(μ-H)B(timMe)(3,5-Me2pz)}(CO)3] (23a) has only been achieved in high yield by reacting the precursor 3a with the sodium salt of the corresponding dihydrobis(azolyl)borate.64 Another versatile and typical organometallic entity is cyclopentadienyl. This chelator acts as a η5-coordinating ligand, occupying three coordination sites. Cyclopentadienyl has been considered a good candidate to stabilize complexes with the fac[M(CO)3]+ (M = Re, Tc) core, aiming at their application in the design of radiopharmaceuticals. The main goal of the studies was to conjugate biomolecules to the cyclopentadienyl or to use the Cp ring as a mimetic of phenyl groups present in relevant biomolecules. In both cases, several difficulties had to be overcome to fully explore Cp ligands in this field, namely the poor solubility and instability of CpH in water, its tendency to polymerize, and high pKa (∼15). The first attempts to obtain half-sandwich complexes of the type fac-[99mTc(η5-Cp-R)(CO) 3 ] were made by Wenzel et al., starting from [99mTcO4]− on the basis of the double ligand transfer approach (DLT) (Scheme 9).65 This approach enabled the synthesis of the desired complexes, but with low yields and under conditions not well-suited for the synthesis of 99mTc

Scheme 7. Reactions of Poly(mercaptoimidazolyl)borate Re(I) Tricarbonyl Complexes with Triphenylphosphine60,62

Scheme 8. Synthesis of Re(I) Tricarbonyl Complexes with Hybrid Poly(azolyl)borates63

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conditions (e.g., pH, presence of tartrate or F− ions), has shown that the presence of tartrate was detrimental to the formation of the carborane complexes, while the presence of fluoride led to the synthesis of the desired complexes (34) in high yield (Scheme 11).73 The role of fluoride is not fully understood, but

Scheme 9. DLT Approach for the Synthesis of fac-[99mTc(η5Cp-R)(CO)3] Complexes

Scheme 11. Aqueous Synthesis of Carborane Tricarbonyl Complexes radiopharmaceuticals on a routine basis. Later on, Katzenellenbogen et al. introduced some improvements on this approach, but the synthesis still needed to be performed in organic solvents.66 The first fully aqueous synthesis of fac-[99mTc(η5-CpC(O)CH3)(CO)3] (30) was described by Alberto and coworkers. As depicted in Scheme 10, complex 30 was prepared by reacting 3a with acetylcyclopentadiene (29), which is more stable, water-soluble, and acidic (pKa ≈ 8) than CpH.67,68 Aqueous organometallic chemistry was also found to be possible with carboxylic and amide derivatives of CpH, having a keto group in an α position. These Cp-based compounds were still rather sensitive, limiting the range of biomolecules to be labeled. Alberto and co-workers further extended the scope of the aqueous chemistry of cyclopentadienyl 99mTc tricarbonyl complexes and showed that Diels−Alder dimerized CpH derivat ives (3 1) react directly with fac-[ 9 9 m Tc(CO)3(H2O)3]+(3a), yielding complex 32 (Scheme 10).69,70 The authors claimed that the retro Diels−Alder reaction involved in the formation of this complex is a metal-mediated process. The dimerized CpH derivatives are much more stable with respect to oxidation or hydrolysis than their monomeric counterparts, and therefore, these results opened new avenues to explore cyclopentadienyl 99mTc tricarbonyl complexes in the labeling of biomolecules. Carboranes play an important role in boron neutron capture therapy (BNCT).71 However, nido-carborane compounds of the [R2C2B9H9]2− type can act also as compact η5-coordinating ligands, isolobal with η5-Cp. Carboranes offer improved water stability and a more versatile functionalization with biomolecules. Valliant et al. studied the aqueous chemistry of carborane 99m Tc tricarbonyl complexes and assessed their suitability for designing targeted-specific radiopharmaceuticals. These authors verified that 3a, obtained from commercially available Isolink kits, did not react to an appreciable extent with carborane ligands.72 A systematic study, using different experimental

99m

Tc(I)

it is thought that its presence inhibits decomposition of the aquo tricarbonyl precursor, during the prolonged heating necessary to obtain the carborane complexes. For certain carboranes bearing sterically demanding substituents and/or biomolecules, the yield of the corresponding rhenium or technetium tricarbonyl complexes can be low, even in the presence of fluoride. Further improvements were achieved using microwave-assisted reactions: in the case of 99mTc, labeling of carboranes using this methodology can be accomplished efficiently (>90%) in a short reaction time.74

5. PERFUSION AGENTS Despite the present interest in target-specific radiopharmaceuticals for nuclear molecular imaging and targeted therapy, there is still scope for the design of alternative and better-performing 99m Tc complexes to replace some established perfusion agents in present clinical use. 99mTc tricarbonyl complexes have recently been preclinically evaluated as perfusion agents for kidney and myocardium imaging. The 99mTc(V) oxo complex 99mTc-MAG3 (35) (Figure 5) is one of the most successful renal radiopharmaceuticals. In the last few decades, it has replaced 131I-orthoiodohippurate (131IOIH), which is no longer in clinical use in the USA and Europe.75 131I-OIH was considered the gold standard for the measurement of effective renal plasma flow (ERPF), but 131I has suboptimal physical characteristics for SPECT imaging and,

Scheme 10. Examples of the Aqueous Synthesis of fac-[99mTc(η5-Cp-R)(CO)3] Complexes

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Figure 5. Structures of 99mTc-MAG3 and 99mTc(I) tricarbonyl complexes with potential as renal radiopharmaceuticals.

being also a β− emitter, delivers high and harmful radiation doses to patients with impaired renal functions. Like 131I-OIH, 99m Tc-MAG3 (35) is mainly excreted by tubular secretion, but its plasma clearance is significantly lower than that of 131I-OIH. It is believed that the presence of a dangling ionized carboxylate and the polar [TcO]3+ group are structural motifs that explain the interaction of 99mTc-MAG3 with the renal tubular transporter. Several hydrophilic Tc(V) oxo complexes, structurally related to 99mTc-MAG3, gave excellent images of the kidney, but none of these complexes presented a clearance higher than that of 131I-OIH.76,77 Recently, Marzilli et al. studied 99mTc(I) tricarbonyl complexes bearing lanthionine (3,3′-thiodialanine, LANH2).78−81 Like MAG-3, LANH2 acts as a N2S donor and presents dangling carboxylates, leading to Tc(I) complexes that were expected to fulfill the structural requisites of renal imaging agents (Figure 5). The reaction of 3a with LANH2 afforded compound 36 (Figure 5). The stereoisomers were evaluated as renal radiopharmaceuticals in healthy volunteers.80 To our knowledge, these two stereoisomers are the only 99mTc(I) tricarbonyl complexes that have been tested in humans. [99mTc(CO)3(LAN)]− (36) had potential as a renal radiopharmaceutical, presenting a rapid renal excretion and providing excellent renal images. Nevertheless, the plasma clearance of 36 was still lower than that of 131I-OIH. The chemical identity of each isomer of 36 was assessed by HPLC comparison with the Re congeners.79,80 Later on, Marzilli and co-workers synthesized and evaluated biologically the complex fac-[99mTc(CO)3(NTA)]2‑ (37) with the tridentate nitrilotriacetic acid (NTA) (Figure 5). Like 99mTc-MAG3, this complex is hydrophilic and dianionic, contains a dangling CO2− group, and is obtained as a single and well-defined species. In a rat model, 37 has shown pharmacokinetics, plasma clearance, and renal excretion similar to those of 131I-OIH. These findings led the author to consider 37 a promising 99mTc tubular agent for the measurement of ERPF in humans. So far, no studies in humans have been reported for fac-[99mTc(CO)3(NTA)]2‑ (37).81 Nuclear cardiology is a very important and noninvasive tool for the clinical evaluation of patients with known or suspected coronary artery disease (CAD), one of the leading causes of death in western countries. The two SPECT radiopharmaceuticals currently available for the clinical management of CAD patients, 99mTc-sestamibi (38) and 99mTc-tetrofosmin (39) (Figure 6), are not ideal, mainly due to their relatively low firstpass extraction and relatively low heart/liver and heart/lung ratios, which may render difficult the clinical interpretation of the heart images due to the interference of the activity retained in adjacent organs.82 Due to the clinical need for better-performing myocardial imaging agents, several research groups searched for lipophilic and cationic 99mTc complexes of efficiency greater than those in

Figure 6. Structures of 99mTc-sestamibi, 99mTc-tetrofosmin and cationic 99mTc(I) tricarbonyl complexes with potential as myocardial imaging agents.

clinical use. Within the tricarbonyl approach, the authors’ and Liu’s groups introduced 99mTc complexes with encouraging biological properties for cardiac perfusion imaging. Liu et al. used a tridentate PNP ligand bearing a pendant crown ether to obtain a 99mTc(I) tricarbonyl complex (40) suitable for myocardial imaging (Figure 6).83 We have focused on a family of 99mTc(I) complexes with tris(pyrazolyl)methane chelators bearing ether substituents at different positions of the azolyl rings.84−87 Of all the complexes, fac-[99mTc(CO)3{HC[3,4,5(CH3OCH2)3pz]]+ (41, 99mTc-TMEOP) (Figure 6) showed the most promising biological profile in mice and rats.85,87 Complex 40 and 99m Tc-TMEOP (41) have shown a significantly fast and stable heart uptake, together with a liver clearance faster than those of 99mTc-sestamibi and 99mTctetrofosmin, as exemplified by 99mTc-TMEOP in Figure 7. Together, the results indicate that these 99mTc(I) tricarbonyl complexes may improve the diagnostic accuracy of CAD and so

Figure 7. Representative SPECT image analysis at 40 min after administration: (A) 99mTc-TMEOP; (B) 99mTc-sestamibi; (C) 99mTctetrofosmin. Reproduced by permission of John Wiley and Sons. 5700


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and Tc tricarbonyl complexes functionalized with arylpiperazines were reported,35,59,67,68,94−96 based on anchor ligands such as dihydrobis(mercaptoimidazolyl)borates (43), Cp derivatives (44), and carboranes (45) (Figure 8). The metal fragment in 43 is stabilized by a dihydrobis(mercaptoimidazolyl)borate containing two pharmacophores, to evaluate how the so-called bivalent approach would improve the pharmacological profile of the molecules.59,94 Organometallic [4 + 1] Tc(III)/Re(III) complexes with isonitrile coligands carrying aryl-piperazines were also evaluated. Several of these complexes presented excellent nanomolar or subnanomolar affinities (IC50 values) toward the 5-HT1A receptors, when linkers of appropriate length were used to attach the pharmacophore to the chelator backbone. However, the [4 + 1] complexes displayed a moderate selectivity for the 5-HT1A receptor and presented nanomolar affinity for αadrenergic receptors.35 This cross reactivity has also been found for the carborane derivative 45.95 Bidentate dithioether ligands bearing tropane derivatives have also been applied in the design of radioactive probes for SPECT imaging of the dopamine transporter (DAT), which is depleted in the striatal region of the brain of Parkinson’s patients. The incorporation of the organometallic core did not compromise the specificity of the tropane moiety toward the DAT.97 Like Parkinson’s disease, Alzheimer’s is a neurodegenerative disorder for which the in vivo targeting of specific biomarkers may allow early diagnostics and monitoring of novel treatments. In Alzheimer’s, for example, A-β peptides form fibrillar deposits in amyloid plaques that invade the brain’s regions of memory and cognition, before spreading to other areas. Important advances have already been made in PET imaging of A-β lesions, but progress is much more mitigated in the case of SPECT imaging, particularly using 99mTc complexes.98 Pirmettis et al. described fluorescent [2 + 1] Re(I) tricarbonyl complexes with a curcumin derivative acting as a bidentate (O,O) ligand and with imidazole or isocyanocyclohexane as unidentate coligands. These complexes bind in vitro to A-β plaques of human post-mortem AD brain sections, as exemplified for 46 in Figure 9.99 So far, no in vivo studies have been reported for the 99mTc congeners.

deserve to be further evaluated in larger animals and humans as myocardial perfusion agents.

6. TARGET-SPECIFIC COMPLEXES Advances in proteomics and genomics have significantly improved the understanding of molecular alterations underlying different diseases, such as the changes in expression and functionality of different genes and/or proteins that emerged as potential targets for in vivo molecular imaging and/or therapeutic purposes.88−92 Among others, antigens, membrane receptors, and enzymes are clinically relevant targets, since they are involved in pathological processes, being in most cases overexpressed or upregulated in comparison to endogenous expression levels. The noninvasive imaging of such alterations with target-specific organometallic radiopharmaceuticals can contribute to the monitoring of disease onset and progression, follow-up of innovative therapies in patients, or preclinical models and dosage optimization of novel drugs. Targeting of Central Nervous System. The design of radioprobes to recognize central nervous system (CNS) receptors and transporters is of great interest, since their density is altered in different psychiatric and neurological disorders. In this field, the best results have been obtained with small and lipophilic organic molecules labeled with PET radionuclides, e.g. 11C and 18F. However, the unique advantages of 99mTc for SPECT imaging prompted several groups to investigate 99mTc complexes functionalized with CNS receptor ligands, using Tc(I) tricarbonyl compounds. The design of this type of complex is challenging, since the metal atom must be incorporated in small-sized and moderately lipophilic compounds, suitable to cross the blood−brain barrier (BBB) and selectively recognize the putative receptors. The compact and lipophilic character of the fac-[99mTc(CO)3]+ core indicated potential for this application. Alberto el al. described one of the first examples of a CNS receptor ligand, labeled with the tricarbonyl fragment using a bidentate Schiff base bifunctional chelator bearing an aryl-piperazine derivative (42), for the targeting of central 5-hydroxytryptamine (5-HT1A) receptors (Figure 8).93 This subtype of receptors is implicated in major neuropsychiatric disorders such as schizophrenia, anxiety, and depression. The Re congener of 42 demonstrated a high affinity and very good selectivity for the 5-HT1A receptor.93 Other Re

Figure 9. Fluorescence microscopy image of an Alzheimer’s diseased brain section, stained with complex 46.99 Reproduced by permission of the American Chemical Society.

Targeting of Estrogen and Androgen Receptors. Hormone-dependent receptors are attractive targets for molecular imaging on the basis of their up- or downregulation in diverse diseases. The estrogen receptor (ER) is overexpressed in two-thirds of breast cancer cases and also in osteoporosis and ovarian cancer.100 The androgen receptor

Figure 8. ORTEP representation of piperazine-containing organometallic complexes.67,93−95 Reproduced by permission of the Royal Society of Chemistry and the American Chemical Society. 5701


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Figure 10. Examples of tricarbonyl complexes for ER targeting.

(AR) has clinical significance in primary and metastatic prostate cancers (PCa), where it is overexpressed.101 A large number of ER-targeted radioprobes have been investigated, including those based on 99mTc(I) tricarbonyl complexes. In general, these complexes were stabilized with tridentate chelators of the type pyridin-2-yl hydrazine, pyrazolyl-diamine, pyridinyl-aminocarboxylic acid, cyclopentadienyl, and carboranes all functionalized for ER targeting, as exemplified in Figure 10.102−107 The most promising of these complexes, the 99mTc(I) estradiol pyridin-2-yl hydrazine derivative 47, has been evaluated in mice bearing MCF-7 and in primary human endometrial tumors. Receptor-mediated uptake in the estrogen receptor expressing tumors was observed.102 However, in vivo studies have shown a high nonspecific uptake in the liver. Jaouen et al. proved that cyclopentadienyl Re(I) tricarbonyl complexes bearing estradiol and nonsteroidal OH-tamoxifen (OH-Tam) derivatives (e.g., complex 49, Figure 10) can act as selective estrogen receptor modulators (SERMs) and, therefore, may also be helpful in designing radioprobes for ER imaging.104,105 Katzenellenbogen et al. have shown that related [ArCpReI(CO)3] complexes are also potent binders of the ER. However, as mentioned before, the synthesis of the 99mTc congeners requires DLT reactions and/or high temperatures, hardly compatible with their routine application in radiopharmaceutical chemistry.106 Seeking to overcome these disadvantages, and taking into consideration that carboranes are nonclassical bioisosteres of aryl rings, Valliant’s group evaluated a series of mono- and diaryl Re and 99mTc metallocarboranes as a new class of agents for ER imaging.107 The metalation of several of these carborane ligands with the fac-[Re(CO)3]+ core led to a significant decrease in their affinity for ERα and ERβ. The replacement of one of the CO ligands by NO+ led to a neutral nitrosyl dicarbonyl Re complex (50) (Figure 10), with a pronounced improvement in the affinity for both receptor subtypes.107 To date, no in vivo studies with the 99mTc(I) congener have been reported. AR targeting has been much less explored than that of ER, despite its relevance as a biomarker of prostate cancer. Published work is based on the use of cysteine, histidine, and Cp ligands functionalized with flutamide, a potent nonsteroidal antiandrogen. These conjugates were labeled with fac-[M(CO)3]+ (M = Re, 99mTc) (e.g., 51 and 52, Figure 11).108,109

Figure 11. Examples of tricarbonyl complexes for AR targeting.

The Cp-based complex 52 showed some uptake by the prostate but was not AR mediated. Like the cysteine congener, the flutamide-containing histidine 99mTc(I) tricarbonyl complex 51 presented a modest in vitro binding to the androgen-positive DU-145 prostate cancer cell line. Targeting of Metabolic Pathways. Several research groups have been involved in the labeling of folic acid or vitamin B12 derivatives with the fac-[99mTc(CO)3]+ core, looking for SPECT radioprobes for tumor detection. Folates and folic acid in their oxidized form are vitamins of the Bcomplex group that play a key role in cell survival and metabolism. The high requirement for folic acid by some cancer cells led to consideration of the folic receptor (FR) as a potential biomarker for tumor detection.110 Vitamin B12 belongs to the same group and also undergoes increased demand from rapidly proliferating tumor cells. The first labeling of a folic acid derivative with fac[99mTc(CO)3]+ used DTPA as bifunctional chelator. Although not the ideal chelator for this core, DTPA afforded a unique and well-defined hydrophilic complex, but with an undefined coordination mode (e.g., N3 vs N2O). The tumor uptake of this [99mTc(CO)3DTPA-folate] complex in a FR-positive tumor xenograft was partially blocked by the coadministration of folic acid. This showed a FR-mediated uptake, although with a fairly high nonspecific component.111 Later, Schibli et al. reported a series of folate-containing 99mTc(I)/Re(I) tricarbonyl complexes using iminodiacetic acid (IDA), picolylamine monoacetic (PAMA), bis(pyridyl)amine, and histidine chelating systems.112−116 Different linkers were used to couple the folate pharmacophore to the BFCAs (Figure 12). Preclinical evaluation of these complexes led to the consideration of a specific in vitro and in vivo uptake in FR-positive human tumor 5702


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microscopy.117 Elsewhere in this special issue the area of organometallics in fluorescent cell imaging is reviewed. Vitamin B12 (cobalamin) has also been labeled with the fac[99mTc(CO)3]+ core using different tridentate chelators (e.g., PAMA, cysteine, or histidine derivatives) or the so-called [2 + 1] approach.45,118−123 The tridentate chelators were conjugated to the 2′-OH function of the ribose or at the b- and the d-acid in the corrin core.45,118,119,121−123 As shown for 56 in Figure 13, in the [2 + 1] approach the cobalamin was coordinated to the fac-[99mTc(CO)3]+ core through a Co−CN bond.120 This research led to different [99mTc(CO)3]-cobalamin complexes that have shown, in general, a significant tumor uptake in B16F10 melanoma tumor bearing mice.45,118,122 As discussed for the folate derivatives, the major drawback of [99mTc(CO)3]cobalamin complexes was their high retention in nontarget organs, mainly kidney and liver. The uptake and transport of vitamin B12 in humans is highly complex. It involves at least three transport proteins (the intrinsic factor (IF), transcobalamin I (TCI), transcobalamin II (TCII)) and the corresponding receptors.124 The highest level of the TCII receptor in human tissues is observed in the kidney, which may explain the high kidney uptake of the cobalamin derivatives.125 Alberto et al. showed that the length of the spacer used to link the BFCA to the b-acid position of cobalamin strongly influences the binding affinity to TCII, when PAMA was the BFCA.122 Interestingly, the complexes with shorter linkers (n = 2−4) lost their binding ability to TCII, while those with longer linkers (n = 5, 6) did not. However, independent of the linker, all [99mT(CO)3-PAMA]-cobalamin complexes recognized the other cobalamin transport proteins: i.e. IF and TCI. Biodistribution studies using tumor-bearing mice have shown that complex 57, with a butylenic linker (n = 4), still has a significant tumor uptake despite the remarkable reduction of the kidney uptake. Blockade experiments suggested a TCI-mediated tumor uptake for 57, while the low accumulation in the kidney most probably results from its low binding to the TCII. Further studies are needed to fully clarify this biological behavior. So far, 57 can be considered the

Figure 12. Examples of tricarbonyl complexes bearing a folate pharmacophore.

cells. The [99mTc(CO)3(histidine-folate)] complex 55 has been considered the most promising as a SPECT probe for imaging FR-positive tumors, due to its significantly higher uptake in tumors, in comparison with the other organometallic radiofolates.116 The main drawback of these folate-containing organometallic complexes is high kidney uptake, as a consequence of FR expression by the proximal tubule cells. More recently, Zubieta et al. described a folate conjugate of Re(I) using a tridentate quinolone-based ligand that conferred fluorescent properties on the respective organometallic bioconjugate. The internalization of that complex in a cisplatin-resistant human ovarian cancer cell line, overexpressing the folate receptor, was visualized by confocal fluorescence

Figure 13. Examples of vitamin B12

99m

Tc(I) tricarbonyl complexes. 5703


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FDG is limited, particularly to detect certain brain tumors or to discriminate between tumoral lesions and inflammatory processes.143 The uptake of radiolabeled amino acids in proliferating tumor cells may reflect an increased protein synthesis rate or simply an enhanced transport across the cell membrane. For instance, the neutral amino acid transporter LAT1 has been found to be highly expressed in nearly all tested tumors and tumor cell lines.144 In recent decades, a number of amino acids (e.g., 11C-methionine, 11C-tyrosine, and 123Iiodomethyltyrosine) sharing a common affinity for the LAT1 transporter have been successfully used for PET or SPECT imaging of human tumors.143 LAT1 can transport a variety of neutral and lipophilic amino acids. Its molecular recognition is less restrictive than that of GLUT-1. Alberto et al. introduced a series of complexes with the fac-[M(CO)3]+ core (M = Re, 99m Tc) stabilized by the tripodal chelator 2,3-diaminopropionic acid functionalized with a pendant glycine group, linked at the α-C or at the terminal amine of the tripod (Figure 15).46 The in

radiolabeled B12 derivative with the most promising profile for tumoral detection, showing that the elimination of TCII affinity may be a good strategy to improve the in vivo performance of this type of probe. Radiolabeled carbohydrates and amino acids can be useful metabolic tracers for molecular imaging applications and/or targeted radiotherapy. However, their labeling with radiometals, such as 99mTc and 186/188Re, is complicated by their small size, and most of the clinically relevant carbohydrate or amino acid based radioactive probes are labeled with 18F or 11C.126 The glucose derivative FDG (2-deoxy-2-[18F]-D-glucose) (58) (Figure 14) has been in use for several decades to image

Figure 14. Examples of carbohydrate-containing M(I) tricarbonyl complexes. Figure 15. Tricarbonyl complexes bearing pendant amino acid groups (M = Re, 99mTc).

glucose metabolism in oncology, neurology, and cardiology. Worldwide it is the most successful PET radiopharmaceutical.126 Significant efforts have been devoted to label carbohydrates with the fac-[M(CO)3]+ (M = Re, 99mTc) core, searching for metabolic SPECT tracers as an alternative to FDG.127 Such compounds have been prepared using bidentate (e.g., aliphatic diamines or bipyridine) or tridentate (e.g., IDA, PAMA, histidine derivatives) Werner-type ligands or cyclopentadienyls.128−141 Coupling of the sugars to the chelating entities has been carried out using different linkers based on the formation of O-glycosidyl, N-glycosidyl, or S-glycosidyl bonds.138,139 As an example, Figure 14 presents compounds with IDA (59/59a) and cyclopentadienyl (60/60a) ligands. Some of the carbohydrate-containing 99mTc complexes were evaluated in tumor-bearing mice and their uptake studied in vitro in tumor cell lines.136−138,141,142 Moderate uptake was observed but, in general, was lower than that obtained with FDG. The most disappointing output of these studies was that none of the evaluated complexes was able to be transported by GLUT-1 as a carbohydrate analogue. In a few cases the interaction of Re complexes with hexokinase (HK) was examined in vitro, but the results indicated that they were not substrates of HK.134−138,142 However, some of the Re complexes inhibited the enzyme with inhibition constants (Ki) in the milimolar range, as for example 59 and 60 containing 2deoxyglucose or 2-aminoglucose derivatives conjugated to the chelators through the C-2 position of the sugar.135,136,142 Another relevant target for metabolic tumor imaging is protein metabolism, which can be targeted with radiolabeled amino acids. Radiolabeled amino acids are expected to be particularly advantageous in situations where the usefulness of

vitro evaluation of the Re complexes using tumor cells overexpressing LAT1 has shown that the complexes functionalized at α-C (61−63) are actively transported and recognize LAT1, with Ki values spanning between 308 and 1100 μM. In contrast, complex 64 functionalized at the terminal amine lost completely its affinity for LAT1. These findings showed that, in addition to size, the topology of the complexes is also an important issue in their interaction with the LAT1 transmembrane receptor. Despite these encouraging results, no biological evaluation has been reported for the 99mTc congeners, and their value as SPECT probes remains unproven. Targeting of Enzymes. The authors have also studied pyrazolyl-diamine Re(I) and 99mTc(I) complexes bearing Larginine derivatives for targeting nitric oxide synthase (NOS) in vivo (Figure 16).145−147 NOS is the eukaryotic enzyme

Figure 16. Examples of L-arginine-based tricarbonyl complexes. 5704


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Figure 17. Synthesis and ORTEP representation of complex 67.153 Reproduced by permission of the American Chemical Society.

anilinoquinazoline type have been labeled with 99mTc using the “4 + 1” mixed-ligand system [Tc(NS)3(CNR)] and the organometallic fac-[99mTc(CO)3]+ core.155,156 In the “4 + 1” approach the quinazoline fragment was introduced through the monodentate isocyanide coligand, 155 while that in the tricarbonyl approach was appended at the imine nitrogen of a bidentate and neutral pyridine-2-methylimine chelator or at the central nitrogen atom of monoanionic N 2 O chelators containing pyrazolyl or pyridine coordinating groups. In vitro studies have shown that the Re complexes inhibit EGFR autophosphorylation, but no in vivo studies have been reported. Targeting of Melanoma with Benzamide Derivatives. Benzamide derivatives are another class of small organic molecules with relevance for in vivo targeting of melanoma and its metastases. Detection of melanoma, a cutaneous neoplasm known for its high aggressiveness, still presents an important challenge in nuclear medicine. Early diagnosis of melanoma and its accurate follow-up can decrease mortality and provide the best chance for optimal clinical management. Encouraging results have been reported for radioiodinated benzamides, which in some cases have undergone clinical trials.157 Taking advantage of the structural similarity of cyclopentadiene and benzene rings, Alberto et al. attempted to mimic benzamides using cyclopentadienyl-based 99mTc complexes.158 The reported results were encouraging in terms of tumor uptake in B16F1 melanoma-bearing mice, but the pharmacokinetics of these Cp-based complexes was not very favorable. The authors’ group has studied an enlarged family of neutral and cationic 99m Tc tricarbonyl complexes with tridentate N3-, N2O-, and N,S,O-donor ligands bearing benzamides or their fragments as melanin binders. In general, these complexes displayed moderate to high in vitro affinity for synthetic melanin, but their tumor uptake in melanoma-bearing mice was lower than expected.159−161 Bone Targeting. Bisphosphonates (BP) are a class of small molecules that are highly important in the treatment of osteoporosis, where they inhibit osteoclatic bone resorption. The osteotropic properties of BPs prompted their labeling with 99m Tc and 186/188Re for bone scintigraphy and therapy, respectively. Some of these compounds are in clinical use, despite their uncertain molecular structure, poor in vivo stability, and low specific activity. Such drawbacks fueled the development of new bone-seeking radiopharmaceuticals, some of them based on the tricarbonyl core.162−165 Pamidronate and alendronate have been labeled with the fac-[99mTc(CO)3]+ core using pyrazolyl diamine162,163 or dipicolylamine (DPA)164 chelators (Figure 18). The resulting 99mTc complexes (e.g., 68a and 69a) were chemically identified by HPLC comparison with their corresponding Re surrogates, which were fully characterized. Such characterization confirmed the formation of well-defined complexes displaying a tridentate coordination of the nitrogen donor chelators and the presence of pendant BP

responsible for the endogenous catalytic oxidation of L-arginine to L-citrulline and nitric oxide (NO), a key mammalian signaling mediator in several physiological processes. Overproduction of NO by iNOS has been linked to many diseases, particularly cancer.148 In vitro studies with Re complexes have shown that the affinity to iNOS of inhibitor-containing conjugates is less affected upon metalation than for the substrate-containing compounds. The Re complexes bearing guanidino-substituted analogues of L-arginine retained considerable inhibitory action and are the first examples of organometallic complexes inhibiting the iNOS.145,146 Remarkably, a Nω-nitro-L-arginine containing Re complex (65) (Figure 16) showed a Ki value of 6 μM, comparable to that of the nonconjugated inhibitor (Ki = 3 μM). Complex 65 also suppressed NO biosynthesis in lipopolysaccharide (LPS)treated macrophages, but the 99mTc congener (65a) did not show any significant uptake in most tissues of LPS-treated mice in comparison to a control group. However, the related complex 66a (Figure 16) showed an enhanced uptake in the LPS-treated animal model, due most probably to iNOS upregulation.146 These results indicate that this family of complexes may hold promise for the design of SPECT probes for in vivo targeting of iNOS. As discussed above for NOS, in vivo imaging of specific enzymes using the respective enzymatic substrates/inhibitors may provide significant information about a variety of diseases associated with their abnormal expression. For instance, significant efforts have been made to obtain Re(I)/Tc(I) organometallic complexes targeting human thymidine kinase (hTK), a cytosolic enzyme with a pivotal role in the synthesis of DNA, which is overexpressed in highly active cells under proliferation: e.g., cancer cells. Schibli’s group synthesized a variety of 99mTc(I) and Re(I) tricarbonyl complexes containing 2-deoxy-thymidine derivatives, using IDA, cysteine, or triazolebased BFCAs conjugated at the C3′, C5′, or N3 position of the nucleoside scaffold.149−152 The Re complexes have been fully characterized, including by X-ray diffraction analysis, as shown for 67 in Figure 17. Their evaluation with the isolated enzyme has shown that the complexes containing the nucleoside functionalized at C5′ displayed, in general, a moderate inhibitory action against hTK, while those functionalized at the C3′ or N3 position acted as hTK substrates.150,153 However, the 99mTc congeners showed a poor uptake in human tumor cell lines. The epidermal growth factor receptor (EGFR), a transmembrane protein of the tyrosine kinase receptor family, is overexpressed in certain cancer cells, such as those of breast and lung carcinomas.154 Several therapeutic approaches are in clinical use or under evaluation for the suppression of tumor growth by inactivation of EGFR signaling. These focus on the use of quinazoline derivatives that act as selective tyrosine kinase (TK) inhibitors. Several TK inhibitors of the 45705


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the fatty acids (71) (Figure 19).169 In vitro studies using isolated Langendorff perfused rat hearts demonstrated that this complex was recognized, transported, and metabolized, as a long-chain fatty acid analogue for energy production in the myocardium. A large number of [4 + 1] mixed-ligand 99mTc(III) complexes, such as 72 (Figure 19), were also prepared and evaluated in vitro and in vivo as fatty acid analogues.36,170,171 The Re congeners were also reported and full characterized, including by X-ray structural analysis (e.g., 73, Figure 19). Some of these [4 + 1] mixed-ligand 99mTc(III) complexes have exhibited high extraction rates in isolated perfused rat hearts, surpassing considerably that of [123I]IPPA. However, biodistribution studies of these organometallic complexes in rat indicated that their heart/liver ratios were not favorable. Targeting of Tumors Using Peptides and ProteinContaining Complexes. Peptides, peptide nucleic acids, antibodies, nanobodies, and affibodies have also been labeled with the fac-[99mTc(CO)3]+ core, searching for SPECT probes suitable for the detection and management of a variety of tumors. These studies have used the bifunctional chelator approach or direct labeling, profiting in the latter case from the high affinity of fac-[M(CO)3]+ (M = Tc, Re) for histidine. This affinity has been recently corroborated by Ziegler et al., showing that fac-[Re(CO)3(H2O)3]+ reacts with hen egg white lysozyme (HEWL), forming an adduct with Nε2 of the His15 imidazole ring (Figure 20).172,173

Figure 18. BP-containing M(I) tricarbonyl complexes for bone targeting.

groups for a more efficient binding to the bone surface. The congener 188Re-DPA-alendronate (69b) (Figure 18) was also reported. The biological evaluation of these BP-containing 99m Tc(I) organometallic complexes in mice has shown a bone uptake comparable to that of the compound in clinical use (99mTc-MDP) with a favorable and fast clearance from most soft tissues. Most relevantly, preclinical studies indicated that 69b is superior to 188Re-HEDP, presenting greater accumulation in regions of high metabolic activity and displaying an enhanced resistance toward in vivo oxidation to perrhenate.165 Altogether, these results clearly indicate that these Tc(I)/ (Re(I) tricarbonyl complexes are a class of complexes with great potential as bone-targeting radiopharmaceuticals for diagnostic or therapeutic applications in nuclear medicine. Fatty Acid Derivatives for Myocardium Imaging. In the search for specific radiopharmaceuticals for nuclear cardiology, as an alternative to perfusion agents in clinical use, several organometallic complexes bearing fatty acids have been synthesized and evaluated. Long-chain fatty acids are the main source of energy in normal myocardium, and alterations in their metabolism are considered indicators of illness, such as ischemic heart disease and cardiomyopathy.166,167 On the basis of 15-(p-[123I]-iodophenylpentadecanoic acid ([123I]IPPA) (70) (Figure 19), a nonmetalated radioprobe in clinical trials for imaging of myocardial metabolism,168 Arano et al. synthesized and evaluated biologically a cyclopentadienyl 99m Tc(I) tricarbonyl complex conjugated at the ω position of

Figure 20. Ribbon diagram of hen egg white lysozyme (HEWL) with Re(CO)3(H2O)2+.172

Table 2 summarizes the peptides that have been labeled with organometallic fragments and their potential application and labeling methodologies. The present paper only refers briefly to this type of work, given that a review on radiolabeled peptides has been recently published.16 With the tricarbonyl core, peptides were labeled using mainly tridentate chelators combining a N-heterocyclic donor group (e.g., histidine, pyrazole, and pyridine) with aliphatic amine and carboxylic acid coordinating functions. Among them, those based on bis(pyridylmethyl)amine and bis(quinolylmethyl)amine ligands functionalized with a pendant N-α-Fmoc-Llysine, designated as single amino acids chelate (SAAC), allow a highly flexible functionalization of the peptides. The SAAC can be introduced at any position of the peptide backbone, and its use is compatible with solid-phase peptide synthesis (SPPS) (Figure 21). The SAAC has been successfully explored for the 99m Tc labeling of the fMLF peptide,202−204 a cyclic peptide for

Figure 19. (A) Examples of 123I or 99mTc-labeled fatty acids. (B) ORTEP representation of a [4 + 1] Re(III) complex functionalized with a fatty acid at the NS3 chelator and with PPh3 as monodentate coligand.170 Reproduced by permission of the American Chemical Society. 5706


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Table 2. Examples of Bioactive Peptides Labeled with Organometallic Fragments amino acid sequence Arg-Gly-Asp-DPhe-Lys (RGDf K)

target integrin avβ3 receptor

diagnosis of rapidly growing and metastatic tumors

cyclo(Arg-Gly-Asp-DTyr-Lys) (cRGDyK)

βAla-Nle4-cyclo[Asp5-His-DPhe7-Arg-Trp-Lys10]-NH2 (βAlaMTII) Ac-Nle4-Asp5-His-DPhe7-Arg-Trp-Gly-Lys-NH2 (NAPamide) Arg-Pro-Arg-Tyr-NH2 ([Phe7,Pro34]-NPY)

DAsp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH2 (CCK8) His-DPhe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-LeuAib-Val-Lys-Lys-Tyr-Leu-NH2 (VD4) His-ACP-DPhe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-LysGln-Leu-Aib-Val-Lys-Lys-Tyr-Leu-NH2 (VD5) Lys(sha)-βAla-βAla-Gln-Trp-Ala-Gly-His-Cha-Nle-NH2 (BBS-42)

melanocortin type 1 receptor (MC1R)

diagnosis of melanoma and metastases

neuropeptide Y1 receptor (Y1R)

diagnosis of breast cancer

cholecystokinin 2 (CCK2)/ gastrin receptor vasoactive intestinal peptide receptor (VPACR)

gastrin-releasing peptide receptor (GRPr)

DPhe-DLeu-DMet-formyl (fMLF)

DLeu-DVal-DPhe-DPhe-DAla-DGly a

histidine174,175 PADA175 pyrazoyldiamine176,177 isocyanide [4 + 1] fashion176 pyrazolyldiamine178−184

histidine185

PADA186 diagnosis of medullar thyroid carcinomas and histidine187 small-cell lung cancers. diagnosis of VPACR-expressing tumors histidine188

diagnosis of breast, prostate, pancreatic, and small-cell lung cancer (SCLC)

Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2 (BBN[7-14])

Arg-(NMe)-Arg-Pro-Dmt-Tle-Leu-OH (NT-XIX)

liganda

application

neurotensin receptor (NTR1) formyl peptide receptor (FPR)

diagnosis of Ewing’s sarcoma, meningiomas, and SCLC imaging of inflammatory processes

β-amyloid plaque

diagnosis of Alzeimerś disease

histidine189−191 pyrazolyldiamine192,193 DTMA194 2,3-diaminopropionic acid193,195 isocyanide [4 + 1] fashion196,197 histidine198−201 bis(pyridylmethyl) amine202,203 bis(quinolylmethyl) amine204 bis(pyridylmethyl) amine205

Abbreviations: DTMA, 2-(N,N-bis(tert-butoxycarbonyl)diethylenetriamine)acetic acid; PADA, 2-picolylamine-N,N-diacetic acid.

Figure 21. Example of a peptide-containing Re(I) tricarbonyl complex obtained with the SAAC methodology.204

the urokinase plasminogen activator receptor,206 an insulin analogue,207 and β-breakers peptides (e.g., DLeu-DVal-DPheDPhe-DAla-DGly).205 More recently, Xiong et al. radiolabeled a library of peptides for the targeting of phosphatidylserine using the SAAC approach, aiming at the detection of apoptosis.208 In general, the metalation of bioactive peptides with the fac[M(CO)3]+ (M = Tc, Re) core did not compromise their specificity and targeting ability. However, in several cases the resulting bioconjugates have shown a relatively poor

pharmacokinetic profile, with abdominal accumulation of radioactivity and hepatobiliar excretion. This unfavorable feature was considered to be due to the hydrophobicity of the metal core. However, more recent results have shown that it results from the use of inappropriate BFCAs. For instance, the authors have introduced a cyclic melanocortin analogue labeled with the fac-[99mT(CO)3]+ core using a pyrazolyl-diamine BFCA. The resulting radiolabeled peptide exhibited a very high tumor uptake in B16F1 melanoma tumor bearing mice (among the highest values reported in the literature) but showed an 5707


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radioactive probes for antisense imaging of gene expression.227,228 The clinically relevant 16-mer PNA sequence HAGAT CAT GCC CGG CAT-Lys-NH2, complementary to the translation start region of the N-myc oncogene mRNA, has been labeled with fac-[99mTc(CO)3]+.229−232 Neither the conjugation of the bifunctional chelator nor the introduction of the fac-[M(CO)3]+ (M = Re, 99mTc) fragment affected the recognition of the complementary sequence and the in vitro stability of the respective PNA/RNA duplex.233,234 Recently, Metzler-Nolte et al. have studied the coupling of an azide derivative of the bis(2-picolyl)amide (Dpam) ligand with an alkyne-containing 12-mer PNA oligomer and evaluated the coordination capability of the resulting bioconjugate, DpamPNA, toward the fac-[M(CO)3]+ (M = Re, 99mTc) core.235 However, the corresponding complexes were obtained with extremely low yields, indicating that Dpam is not a suitable ligand to stabilize the tricarbonyl core. Nanocompounds for SLND. The design of nanosized probes for in vivo molecular imaging applications is a growing field. The authors’ group and others have explored tricarbonyl technology to label mannosylated dextran derivatives, with the objective of identifying well-defined radioactive nanocompounds for sentinel lymph node detection (SLND). Such detection is very important for planning the extent of surgery, tumor staging, and establishment of the most adequate therapy. The dextran was functionalized with pyrazolyl-diamine or cysteine for stabilization of the fac-[M(CO)3]+ (M = 99mTc, Re) core (Figure 23).236−238 SPECT/CT studies in mice

undesirable accumulation of activity in the liver and intestine.179 The biodistribution profile of this radiometalated peptide was significantly improved through the introduction of a free carboxylate at the 4-position of the pyrazolyl ring, leading to a 99mTc-labeled melanocortin analogue with a very high and specific tumor-targeting capability (21.7 ± 5.7% ID/g, 1 h post injection) and with minimal retention of activity in the hepatobiliary tract (5.5 ± 0.4% ID/g, 1 h post injection) (Figure 22).209 A new series of lysine-derived SAAC systems

Figure 22. Planar image of a melanoma-bearing mouse injected with a 99m T(CO)3-labeled melanocortin analogue.209

containing polar groups (e.g., carboxylic, ether, and alcohol) have also been synthesized to improve the pharmacokinetics of a 99mTc-labeled octreotide analogue.210 Radiolabeled antibodies have also attracted attention in the nuclear imaging and therapy of cancer. However, probes based on whole antibodies present several disadvantages: in particular, their unfavorable blood clearance. To avoid this drawback, smaller-sized antibody fragments, nanobodies (15 kDa) and affibodies (7 kDa), are possible alternatives, as they commonly present better tumor penetration and faster clearance, providing high tumor to background ratios at earlier postinjection time points.211 The conjugation of hexahistidine (H6) to the N terminus of polypeptides or proteins enables their purification using immobilized metal ion affinity chromatography (IMAC), offering also the opportunity for their labeling using the tricarbonyl approach.212 This hexahistidine-tag technology has been successfully explored for the [99mTc(CO)3]+ labeling of specific nanobodies targeted at the epidermal growth factor receptor (EGFR)213−217 and for vascular cell adhesion molecule-1 (VCAM-1)218 for SPECT imaging of tumors and atherosclerotic lesions, respectively. The labeling of human epidermal receptor type 2 (HER2) specific affibody (ZHER2:342) with fac-[99mTc(CO)3]+ via the H6 tag led to a radioconjugate with a relatively high liver accumulation.219,220 However, replacement of the H6 tag by a more hydrophilic HEHEHE tag has yielded a stable 99mTc-labeled ZHER2:342 with reduced hepatic accumulation.221 Peptide nucleic acids (PNAs) are another class of peptidebased molecules that also have relevance for designing PET or SPECT radioprobes. PNAs are non-natural nucleic acid analogues, in which the sugar−phosphate backbone of DNA/ RNA is replaced by a pseudopeptide chain constituted by N-(2aminoethyl)glycine units.222,223 Thanks to their high selectivity for DNA/RNA, high stability to a wide range of pH, and resistance to enzymatic degradation by nucleases/proteases,224 PNAs have been largely investigated as agents for antisense and antigen therapy.225,226 PNAs have also been investigated as

Figure 23. Mannosylated dextran derivatives.

confirmed that the resulting 99mTc-labeled dextran derivatives accumulate in the popliteal lymph node, allowing its clear visualization (Figure 24). These nanocompounds deserve further evaluation as radiopharmaceuticals for SLND. Cell-Specific and Nuclear Targeting. 99mTc is also an Auger-electron emitting radiometal, and therefore, 99mTc complexes have potential for targeted antitumor therapy. However, the short range of Auger electrons necessitates the accumulation of 99mTc complexes in the nucleus of tumor cells in order to elicit significant DNA damage and therapeutic effect. This requires multifunctional compounds displaying a cellspecific uptake and the ability to target the nucleus. One possibility is to use a bioactive peptide as a targeting vector, with a DNA binder to enhance nucleus internalization and 5708


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25).241−243 These pyrazolyl-diamine complexes rapidly entered the cells and accumulate inside the nucleus. Complex 77a, having the anthracenyl substituent at the 4-position of the pyrazolyl ring, exhibited pronounced radiotoxic effects and induced an apoptotic cellular outcome.241 However, a challenging task would be to design a cell -specific 99mTc complex with the ability to accumulate in the nucleus. To address this, Alberto et al. synthesized the Re(I)/99mTc(I) tricarbonyl complexes 78/78a functionalized with a bombesin (BBN) analogue for receptor-mediated uptake in cells overexpressing the gastrin releasing peptide receptor (GRPr) (Figure 25). Fluorescence microscopy studies of 78 in GRPr-positive PC3 human prostate tumor cells have shown a rapid and specific internalization into the cytoplasm but poor uptake in the nucleus.197 Similar results were reported for [2 + 1] complexes stabilized with an AO-containing 4-imidazolecarboxylate bidentate ligand and having a BBN analogue introduced through the unidentate isonitrile coligand.244 The same authors also hypothesized that the use of a cleavable disulfide linker to couple BBN analogues to [2 + 1] 99mTc(I)/ Re(I) tricarbonyl complexes should release intracellularly the organometallic moiety containing the AO intercalator, leading to a more efficient nuclear targeting. In vitro studies have shown that the disulfide linker was cleaved by glutathione (GSH) at a reasonable rate, but the complexes did not display an enhanced uptake in the nucleus, although they showed a cell-specific uptake.55 The authors have obtained more encouraging results using pyrazolyl-diamine 99mTc(I)/Re(I) tricarbonyl complexes bearing BBN analogues and AO intercalators. From the studied 99mTc complexes, the authors have found that complex 80, containing the GGG-BBN[7-14] peptide (Figure 25), presented a high cellular internalization in PC3 cells and a remarkably high nuclear uptake in the same cell line. As shown in Figure 27, live cell confocal imaging microscopy studies with the Re congener complex 80 have shown a considerable accumulation of fluorescence into the nucleus with uptake kinetics similar to that exhibited by 79. These compounds are the first examples of 99mTc bioconjugates that combine specific cell targeting with nuclear internalization, a crucial issue for the exploitation of 99mTc in Auger therapy.245

Figure 24. SPECT/CT image in mice after subcutaneous injection of the 99mTc-labeled dextran derivative 75 (see Figure 23).239

promote close proximity to DNA. This type of study was pioneered by Alberto et al., who have shown that a trifunctional 99m Tc(I) tricarbonyl complex, containing a pyrene intercalator and a NLS peptide, can reach the nucleus of B16-F1 mouse melanoma cells and induce much stronger radiotoxic effects than 99mTcO4−.240 Alberto et al. demonstrated that a [2 + 1] Re(I) tricarbonyl complex (76) (Figure 25) bearing acridine

Figure 25. M(I) tricarbonyl complexes functionalized with polyaromatic DNA intercalators.

7. CONCLUDING REMARKS AND PERSPECTIVES From all the organometallic precursors recently introduced in radiopharmaceutical chemistry, fac-[M(CO)3(H2O)3]+ (M = Re, Tc) have been the most explored, mainly in the design of radioprobes for SPECT imaging. Since the introduction of these precursors, many research groups worldwide have studied their reactivity with BFCAs of different denticities and donor atom sets. For the first time, 99mTc tricarbonyl complexes with bridging hydrides, cyclopentadienyls, and carboranes have been synthesized in aqueous solution, in quantitative or very high yield. These and other building blocks, anchored by Wernertype ligands, were explored for labeling a huge variety of biomolecules, to obtain new radiotools suitable for diagnostic or therapeutic applications in nuclear medicine. So far, there are no tricarbonyl complexes in clinical use, but very encouraging results have been obtained at the preclinical level for perfusion and target-specific agents. The versatility of the tricarbonyl approach has allowed fine tuning of the pharmacokinetics and biodistribution profile of some compounds, showing that the inherent lipophilic character of the tricarbonyl core can be modulated by the nature of the BFCA. This has been shown to be the case for renal and myocardium perfusion agents,

orange (AO) as a DNA-binding group can target the nucleus of murine B16F1 cells without needing a carrier NLS sequence. Interestingly, the tight binding of 76 to DNA was clearly visualized in dividing PC3 cells (Figure 26).197 However, the radiotoxicity of the 99mTc(I) congener (76a) has not been reported. The authors have also evaluated pyrazolyl-diamine 99m Tc(I)/Re(I) tricarbonyl complexes bearing anthracene (77) and acridine orange groups (79) for DNA interaction (Figure

Figure 26. Complex 76 binding to the nuclear DNA of PC-3 cells during cell division.197 Reproduced by permission of John Wiley and Sons. 5709


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Figure 27. Live cell uptake of complex 80 in PC3 cells, visualized by time-lapse confocal microscopy imaging.245

osteotropic complexes, bioactive radiopeptides, and labeled dextran derivatives, which have emerged as new highperformance radiotools that justify further clinical evaluation. For many years, considerable attention has also been devoted to the synthesis and biological evaluation of tricarbonyl complexes functionalized with small biomolecules, such as carbohydrates, amino acids, and enzyme substrates/inhibitors. For these types of biomolecules it has proved more difficult to avoid interference of the organometallic fragments with their biological performance, and reported bioconjugates with clinical potential are scarce. Nevertheless, the unique features of Re(I)/Tc(I) tricarbonyl complexes allow for the innovative design of chelators to mimic structural motifs of small biomolecules, which is a crucial issue for the improvement of biospecificity and pharmacokinetics. Intense research related to tricarbonyl precursors has shown that organometallic chemistry is compatible with the experimental conditions used in radiopharmaceutical chemistry. The versatility of this core has opened new avenues in the field, which shows great potential for further exploration.

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of Lisbon, where she teaches several courses, including the Master's Course “Biomedical Inorganic Chemistry: Diagnostic and Therapeutical Applications”, being responsible for its organization and coordination.

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ACKNOWLEDGMENTS The support of the FCT (PTDC/QUI-QUI/115712/2009), COST CM1105, and COST TD1004 are acknowledged. G.R.M. thanks the FCT for a contract under the “Ciência 2008” program.

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REFERENCES

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AUTHOR INFORMATION

Notes

The authors declare no competing financial interest. Biography

Isabel Santos received a degree in chemical engineering from the Technical University of Lisbon, Lisbon, Portugal, where she also received her Ph.D. and Aggregation in chemistry. She works at the Instituto Tecnológico e Nuclear/Instituto Superior Técnico-Technical University of Lisbon, where she was appointed as Senior Researcher in 1999 and as leader of the Radiopharmaceutical Sciences Group in January 2000. In this multidisciplinary group, Isabel Santos and her coworkers are interested on the design of innovative tools for SPECT and PET molecular imaging and targeted radiotherapy. These metalbased tools are designed to target specifically biomarkers related to cancer and neurodegenerative diseases. Isabel is author or coauthor of more than 180 scientific papers and several invited reviews and is coinventor of some patents. She has supervised 4 Master's students, 12 Ph.D.'s, and 9 Postdoctoral fellows. She is Professor at the University 5710


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Organometallics

Review

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Organometallics

Review

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